Radiotherapy using "non-sealed sources" by way of radiolabeled pharmaceuticals has been employed for several decades. Spencer et al., 1987; Schlom, 1986; Saenger, 1979. Unfortunately, less than a handful of therapeutic radiopharmaceuticals are currently in routine use, as being approved by the FDA. There has been renewed interest in developing new agents due to the emergence of more sophisticated molecular carriers, such as monoclonal antibodies that are capable of selectively targeting cancerous lesions. In addition, the identification of several different radionuclides Volkert et al., 1991; Schubiger and Hasler, 1986; Mausner et al., 1988; Andres et al., 1986 with different chemical properties that have physical decay properties that are desirable for therapeutic application have further spurred development of new agents.
Despite some successes in treatment of specific malignant diseases and increased research and development activities in this area, many problems remain with the use of such treatments. For example, it has been difficult in most cancers to provide acceptable selectivity in radiation doses delivered to target tissues relative to normal tissues. Successful development of new therapeutic radiopharmaceuticals requires improved localization of these agents in target tissues and/or increasing rates of clearance from non-target tissues. In both of these cases, it is imperative that the therapeutic radionuclide remain firmly associated with the radioactive drug in vivo for extended periods of time. These periods of time can extend from a few hours up to several days, depending on the pharmacokinetics and physical half-life of the radionuclide. No single radionuclide can be appropriate in formulating therapeutic agents since different half-lives and the energy of emitted particles is required for different applications [Volkert et al., 1991; Schubinger and Hasler, 1986; Mausner et al., 1988; Andres et al., 1986] thereby making it essential that radiopharmaceuticals with different radionuclides be available.
Therapeutic agents have been primarily labeled with beta-particle emitting radionuclides. Most of the promising radionuclides are produced in nuclear reactors, however, some are accelerator produced. [Volkert et al., 1991; Schubiger and Hasler, 1986; Mausner et al., 1988; Andres et al., 1986]. Several different chelating structures have been employed to maintain the association of these beta emitters with the drug. [Kozak et al., 1989; Hnatowich, 1990; Rao et al., 1988; Deshpande et al., 1990; Washburn et al., 1990]. Many of these structures are not sufficiently stable and most, if not all, do not provide appropriate routes or rates of clearance of radioactivity from non-target tissues. [Meares et al., 1988; Naruki et al., 1990]. Accordingly, there is a delivery of high radiation doses to normal tissues and a reduction of the therapeutic ratio. This lowers the amount of radiation dose that can be safely delivered to a target tissue. Development of new radionuclide that link the radioactive metal to the radiopharmaceutical is necessary. Further, new approaches must be taken in order to identify radio-labeling techniques that produce chelates that are highly stable in vivo but have improved clearance characteristics from normal tissues.
Bi-functional chelating agents have been used to form stable metal complexes that were designed to minimize in vivo release of the metallic radionuclide from the radiopharmaceutical. For example, diethyltriaminepentaacetic acid (DTPA) forms rather stable chelates with a variety of metals. However, coupling of this ligand to monoclonal antibodies by one of its five carboxyl groups resulted in unacceptable in vivo stability with a variety of radionuclides [Parker, 1990]. Linking of this compound by a side group attached to one of the carbon atoms on an ethylene bridging group provides improved stability in vitro and in vivo. The stability characteristics of these compounds are not ideal resulting in poor clearance of activity from certain non-target organs are poor.
Chelating agents based on diamidodithiol and triamidomonothiol backbones are used for forming small and stable hydrophilic complexes with several beta-emitting transition metals. These were developed by Fritzberg and colleagues [1988] for labeled monoclonal antibody products for diagnostic and therapeutic applications. These chelates provide improved clearance characteristics from the liver; however, kidney retention of activity when using Fab or (Fab).sub.2 fragments of monoclonal antibodies labeled with these radionuclide chelates is higher than desirable.
A macrocyclic tetramine-based chelating agent that also has four methylene carboxylate side atoms has been used to form a copper complex that has a high in vitro and in vivo stability when linked to monoclonal antibodies. This chelate was first described for monoclonal antibody bioconjugation by Meares and coworkers. [Deshpande et al., 1990; Washburn et al., 1990] The chelate is rather large. Clearance of activity from non-target organs has not been shown to be more efficient than the chelates mentioned above.
Recently, a mono-hydrazide bifunctional chelating agent has been described that forms somewhat stable .sup.99m Tc complexes. [Abrams et al., 1990a: Abrams et al., 1990b] This particular ligand can be first attached to a protein and then binds to .sup.99m Tc as it is chelated with glucoheptonate in aqueous solutions at or near neutral pH. [Abrams et al., 1990b] Other work with mono-hydrazide ligands with other Tc.sup.v complexes demonstrates the reaction of the monohydrazides at the axial position with Tc.sup.v =O to form similar mondentate linkages. [Abrams et al., 1990a] The in vitro and in vivo stability of these types of chelates has not been adequately described in the literature.
Applicant has synthesized a series of multi-dentate ligands derived from a phosphorous or germanium core utilizing hydrazine groups as arms of these ligands to form small but stable and well defined complexes with transition metal radionuclides. Unlike prior art chelates, these chelates show good stability in both aqueous solutions, serum, and other body fluids.